Designing mutant IL - 12 for modulating IFN - Y Production

Abstract

Cancer is a set of multiple diseases, characterized by uncontrollable abnormal cell growth that has the ability to infiltrate and destroy normal body tissue. Depending on the type and stage of cancer, treatments are given to eradicate the tumor. Chemotherapy is one of the treatments, leads to intense pain, and chances of remission of cancer. So, the recent focus of the researcher is cancer immunotherapy. Recent studies have highlighted the feasibility of using immunomodulatory cytokines such as Interleukin (IL)-2, IL-12, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), and type 1 interferons (IFNs). Out of these cytokines; IL-12 holds the fact that it plays a central role in regulating both innate and adaptive immune responses. The administration of the cytokines is critical for the reason that they have paracrine and autocrine effect, low concentration may not be able to show its effect whereas high concentration leads to toxicity. IL-12 is a heterodimer made up of p35 and p40 subunits, p40 helps in the binding of IL-12 with its receptor and p35 activates the signalling pathway. IL-12 is the most potent cytokine to be used as an immunotherapeutic agent but the administration of multiple doses produces systemic toxicity and hence it is one of the limiting factors in clinical trials. When IL-12 is administered, it stimulates CD8+ T and NK cells which are cytotoxic in nature. These cells stimulate a high level of IFN-γ production that leads to the lethal inflammatory syndrome. To study the signaling of IL-12 and to target the reduced toxic treatment for tumors, we try to mutate the subunits of IL-12. To mutate the p35 subunit SDM (Site Directed Mutator) is used. The structure of IL-12 receptor beta 1 and beta 2 was predicted using different tools like SWISS-MODEL, I-TASSER, PHYRE2, and Robetta. The accuracy of these structures was not optimal and hence the cytokine binding region of the receptor was predicted using gp130 cytokine binding region as a reference structure. The modeled domains were then used to dock the original p35 and p40. The generated p35 mutants were then docked with p40 to observe the interactive residue and p70 formation.It has already been shown that the IL-6/IL-6 receptor alpha complex is similar to the IL-12/1L-12 receptor complex. As both have cytokine binding regions, it helps us to select the docked structures for wild type and mutants with the IL-12 receptor and further it is narrowed down with stability energy. These mutants can be used for in vitro experiments for further studies. Since, the crystal structure is not available, using a different web server such as SWISS-MODEL, I-TASSER, PHYRE2, and Robetta modeling are done for the IL-12 receptors to which both the subunits bind. The wild-type and mutant p35 are docked with the modeled receptor using ClusPro and the common residues were selected. These residues will help in the interaction study of IL-12 with the receptor. We are expecting that these mutations would alter the IFN-γ production and the mutant that produces a low amount of IFN-γ production can be used in cancer immunotherapy.